The action potential of excitable membranes is determined primarily by the fast activation and inactivation of voltage gated sodium (Na) channels, which occur with time constants on the order of milliseconds. More prolonged membrane depolarizations (hundreds of milliseconds) result in a second type of inactivation, recovery from which takes place with time constants on the order of seconds. Interest in the molecular mechanism of this "slow inactivation" has been heightened by recent studies implicating derangements of both fast and slow inactivation in inherited periodic paralyses. The proposed study seeks to define regions of the human skeletal muscle voltage gated Na channel (hSkM1) which participate in slow inactivation. Scanning point mutagenesis of regions previously implicated in slow inactivation will be performed to define the boundaries and specificity of these regions. In addition, we will take advantage of the difference in the extent of slow inactivation between adult cardiac (hH1) and skeletal muscle (hSkM1) Na channels in studies utilizing protein-protein chimeras, to identify novel regions which participate in slow inactivation. Chimeric protein studies will also be used to test the hypothesis that pore-forming segments may be differentially involved in slow inactivation. Finally, the contribution of the S4 voltage sensors in slow inactivation will be examined by assessing the availability of substituted cysteine residues to covalent modification by thiol-reactive reagents in both resting and slow inactivated states. We anticipate that more detailed knowledge of the molecular mechanism(s) of slow inactivation will provide a framework for rational therapeutic strategies for familial periodic paralyses and perhaps other disorders of membrane excitability. The applicant has received both M.D. and Ph.D. degrees, and is currently completing clinical training as a resident in Neurology. He anticipates that the proposed training will enable him to establish himself as an independent researcher within the three year funding period.